This invention relates to synthetic substrates and their use for determining the activities of enzymes, and more specifically, to peptide thioneamides and their use for determining the activities of cysteine proteases.
The determination of specific enzymes in biological fluids, such as blood, tissue homogenates, urine, or the like, is very useful for the diagnosis of certain diseases, e.g. Evered et al., eds. Protein Degradation in Health and Disease (Exerpta Medica, Amsterdam, 1980). Synthetic substrates have been developed and utilized for such determinations, resulting in clinical assay procedures having a high degree of specificity, reliability, and sensitivity. Synthetic substrates have generally been amino acid or peptide derivatives acylated to aromatic amines, the latter becoming fluorimetrically or spectrophotometrically detectable after being cleaved from the amino acid or peptide, e.g. see Lorand, ed.,"Proteolytic Enzymes," Methods in Enzymology, Vol. 80 (Academic Press, New York, 1981). The number and ordering of amino acids in the peptide moiety determines the enzyme specificity of a substrate. Enzyme activity is measured by the amount of the aromatic amine moiety liberated upon hydrolysis of a substrate. Exemplary synthetic substrates are disclosed in U.S. Pat. No. 3,862,011 dated Jan. 21, 1975 to Smith, U.S. Pat. No. 4,294,923 dated Oct. 13, 1981 to Smith et al.; and U.S. Pat. No. 4,505,852 dated Mar. 19, 1985 to Rasnick.
Enzyme specificity is an important criterion for the use of synthetic substrates in evaluating proteolytic activities, particularly in the clinical setting. For example, the accuracy and reliability of diagnostic tests based on measuring protease activities would be increased significantly if synthetic substrates were available which allowed the measurement of a particular protease activity in a biological fluid containing a host of related proteases. That is, such tests would be significantly improved by the availability of synthetic substrates for particular enzymes having little or no cross reactivity with related enzymes present in the same biological fluid.
The serine proteases are perhaps the best understood class of enzymes in their catalytic mechanism, and a number of highly specific synthetic peptide substrates are available for their detection. Cysteine proteases, on the other hand, have not shared the same intensity of interest until recently. During the last decade considerable interest in cysteine proteases has arisen as evidence of their involvement in a number of pathological conditions has become more certain. Cathepsins B, H, and L have been linked to inflammation, e.g. Ostensen et al. Clin. Exp. Immunol., Vol. 54, pgs. 397-404 (1983), and protein degradation, e.g. Sutherland et al., Biochem. Biophys. Res. Comm., Vol. 110, pgs. 332-338 (1983); McDonald et al., Anal. New York Acad. Sci., Vol. 380, pgs. 178-186 (1982); Quinn et al., Biochem. J., Vol. 172, pgs. 301-309 (1978); and Ishura et al., J. Biochem., Vol. 94, pgs. 311-314 (1983). Many tumor cells have been found to secrete a cathepsin B-like cysteine protease which enables the tumor cells to invade the extracellular matrix and to metastasize to secondary sites, e.g. Sloane et al., Cancer Metastasis Reviews Vol. 3, pgs. 249-263 (1984). Unfortunately such studies are limited because currently available synthetic peptide substrates used to assay cysteine proteases are often hydrolyzed by serine proteases, making it difficult to assign the observed activities to cysteine proteases with confidence.
More specific and selective substrates are necessary to improve or create new diagnostic tests based on cysteine protease activities, and to study the specific functions of cysteine proteases in normal and pathological states.